ARTICLE

Overview

Since the seminal work in two-photon 안전한 바카라 사이트 scanning fluorescence microscopy published in 1990 (Denk, et al., 1990), the technique has benefitted from step changes in 안전한 바카라 사이트 technology. These improvements have furthered the penetration of the technique initially from a physics laboratory, to cell biology, disease studies, and advanced neuroscience imaging.

One box, tunable Ti:Sapphire 안전한 바카라 사이트s started this trend around 2001. Some years later, automatic dispersion control was added to the 안전한 바카라 사이트s to optimize the pulse duration at the sample plane of the microscope. As probes excitable at wavelengths longer than the upper limit of Ti:Sapphire 안전한 바카라 사이트s became more mature and efficient, after 2010 안전한 바카라 사이트 companies turned to Optical Parametric Oscillators to address this need for an augmented color palette, deeper imaging, and less photodamage.

In this article, we discuss the next phase in this evolution; the integration of fast power modulation in the 안전한 바카라 사이트 system, and how this enables faster setup time, highest performance, and low cost of ownership.

Requirements for 안전한 바카라 사이트 Power Control in Two-Photon Microscopy.

In its simplest form, continuous control of 안전한 바카라 사이트 power can be achieved by addition of a phase retarding waveplate and polarizing analyser. By rotation of the waveplate, the transmission of the 안전한 바카라 사이트 power through the analyser can typically be changed from 0.2% transmission to ~99%. By motorizing the waveplate this process can be automated to alter the power at the imaging plane in the microscope to equalize focused fluences at different depth frames, for example.

Most modern 안전한 바카라 사이트 scanning two-photon microscopes, however, require a faster modulation speed. For example, for a raster 안전한 바카라 사이트 scanning application where data acquisition should be made in a single direction only, the 안전한 바카라 사이트 must be blanked during the “flyback operation” to avoid unwanted fluorescence excitation or photobleaching. In the case of resonant galvometric scanners, the resulting rise/fall times can be as small as a few microseconds. In this domain, one must consider optical modulation methods.

Electro-optical 안전한 바카라 사이트

An Electro-Optic Modulator (EOM) modulates 안전한 바카라 사이트 power by applying a phase retardation to the beam by use of the Pockels effect. This is where birefringence is induced in a non- centrosymmetric crystal by application of an electric field. As before, a polarizing analyzer is used to complete the modulator setup.

Pockels cells may be configured in a longitudinal excitation geometry, to accommodate larger beams with relatively short crystals. In this case the typical 1⁄2 wave voltage (i.e the voltage required for a 90-degree rotation of the polarization), is in the order of 6 kV, which is difficult to achieve at the speeds and duty cycles required for 2P 안전한 바카라 사이트. Thus, most configurations for imaging adopt the transverse electric field geometry, using longer crystals, which significantly lowers the halfwave voltage. Crystals are generally deployed in 2 or more serial configurations, rotated with respect to each other, to further lower the required switching voltage and compensate for thermal loading effects.

Care must be taken to optimize pulse 안전한 바카라 사이트ntrast (ratio of minimum versus maximum transmitted power) by crystal alignment and offset (bias) voltage adjustment for best image 안전한 바카라 사이트ntrast.

Pockels cells are widely adopted in 안전한 바카라 사이트 microscopy, especially amongst the “home builders” community as they are relatively simply deployed, especially for users requiring only modest powers at popular 안전한 바카라 사이트 wavelengths.

For example, cells based on potassium dideuterium phosphate (KD*P) offer excellent transmission, speed, and contrast characteristics for 2P applications up to approximately 1100nm, and modest 안전한 바카라 사이트 powers. Additionally, KD*P has low group velocity dispersion characteristics, resulting in minimal group delay dispersion (GDD). To this end KD*P Pockels cells are a popular choice when using ultrafast 안전한 바카라 사이트s without dispersion precompensation and limited tuning, such as Ti:Sapphire 안전한 바카라 사이트s.

Acousto-optic 안전한 바카라 사이트

An A안전한 바카라 사이트usto-Optic Modulator (AOM) 안전한 바카라 사이트mprises a transparent crystal or glass onto which a piezoelectric transducer is attached. A radiofrequency (RF) wave applied to the transducer induces an a안전한 바카라 사이트ustic wave that strains the crystal resulting in a refractive index grating. Light travelling through the cell then undergoes Bragg diffraction.

The rise/fall time achievable is proportional to the time the acoustic wave takes to cross the 안전한 바카라 사이트 beam, thus is optimized by reducing the beam’s width in the crystal.

Discrimination and therefore 안전한 바카라 사이트ntrast ratio is defined by both the separation angle (θ_S) between the zero and first diffract안전한 바카라 사이트 orders and the distance to the working plane of interest.

The most common AOM material used in 안전한 바카라 사이트 microscopy is tellurium dioxide, (TeO₂). This material demonstrates excellent diffract안전한 바카라 사이트 efficiency and high-power handling over a wide wavelength range. Maximum transmiss안전한 바카라 사이트 efficiencies are achieved with modest RF powers in the order of 30 dBm.

TeO₂AOMs are usually configured in the Bragg interaction regime which offers best diffraction efficiency into the first order, with higher orders destructively annihilated. Note that to achieve high efficiency with minimal RF power levels, crystal lengths of 1 cm are needed, resulting in non-negligible group delay dispersion (GDD). Considering also the dispersion of other downstream optics and especially the objective lens, AOM-based microscope systems benefit by being partnered with 안전한 바카라 사이트s equipped with dispersion precompensation, in order to maintain the shortest pulses at the sample plane.

Deployment of AOMs for tunable 안전한 바카라 사이트s requires both careful optical and control electronics design. Since the separation angle (θ_s) is dependent on both the RF drive frequency (ie grating period) and the 안전한 바카라 사이트 wavelength, the RF drive frequency must be carefully calibrated to ensure minimal pointing change when tuning the 안전한 바카라 사이트 wavelength. Additionally, the maximum diffraction efficiency is achieved at different RF powers for different wavelengths. The more onerous integration effort resulting from the need to carefully control the RF frequency and power, and manage a relatively large GVD in a tunable imaging system has hitherto limited AOM usage in many home-builder and custom settings, despite the excellent performance characteristics.

Modulation in Widely tunable 안전한 바카라 사이트s

The advent of one-box widely tunable 안전한 바카라 사이트s, in the order of 680-1300 nm and with powers in excess of 2 W, requires a new regime of performance and integration effort for 안전한 바카라 사이트 modulation.

The typically used KD*P Pockels cells display thermal blooming effects at high power, which is deleterious to beam pointing, beam waist integrity, and lifetime. Longer wavelengths further present higher drive voltage and contrast challenges. Lithium Tantalate is a viable EOM material for wider tuning, however the group delay dispersion of commercial units is higher than the correctable range of dispersion-compensated 안전한 바카라 사이트s, thus resulting in longer pulses and reduced peak power, detrimental to efficient imaging.

As previously discussed, despite having potential 안전한 바카라 사이트st and performance benefits, AOM-based solutions require a high degree of optical design and electronics 안전한 바카라 사이트ntrol expertise to deploy, often not readily available in many bioimaging facilities. That said, AOM solutions are 안전한 바카라 사이트mmercially available as an integrated solution from some micros안전한 바카라 사이트pe vendors.

In 2017, Coherent recognized that both users and the microscope industry would benefit from a turn-key solution integrating the AOM modulation with the 안전한 바카라 사이트 sources. Building on the expertise gleaned from integrated AOM solutions in industrial ultrafast machining 안전한 바카라 사이트s, Coherent developed Total Power Control (TPC) – as a fully integrated option for the Chameleon Discovery 안전한 바카라 사이트.

Total Power Control, available on Chameleon Discovery NX, provides high contrast (1000:1) and high speed (<1 μs rise time) 안전한 바카라 사이트 across a full octave tuning range of 660 nm to 1320 nm in a hands-free automated package.

All the taxing requirements for RF frequency and power calibration and adjustment are programmed internally to the 안전한 바카라 사이트, so all the user or microscope integrator needs to provide is the set wavelength and power level required.

Since AOMs are very 안전한 바카라 사이트st-effective, the fixed wavelength 1040 nm output of Chameleon Dis안전한 바카라 사이트very NX TPC is also equipped with its own, dedicated AOM and driver.Power can be 안전한 바카라 사이트nveniently 안전한 바카라 사이트ntrolled by either serial/USB 안전한 바카라 사이트mmand or by fast analog 안전한 바카라 사이트ntrol input.

Future trends

As the scope of 안전한 바카라 사이트 imaging techniques drives further into OEM and preclinical applications, the demand for single wavelength, cost-effective femtosecond sources is growing. The Axon series of compact ultrafast sources addresses perfectly these requirements.

From product concept stage, TPC capability was integrated into the Axon design to simplify deployment into new microscope designs and applications. This brings ultimate integration convenience for applications where the 안전한 바카라 사이트 microscope system is part of a movable diagnostic, clinical or high-throughput screening device rather than a pure research instrument.

In cutting-edge neuroscience research, high-power 안전한 바카라 사이트s are playing a key role in all-optical in- vivo imaging techniques, using optogenetic stimulation (Yuste, 2012). Multiple tens of watts of 안전한 바카라 사이트 power are split with spatial light modulators (SLMs) into individual beamlets able to individually address tens or hundreds of neurons. This method of optical control requires short and tailorable burst of pulses. High-power fiber 안전한 바카라 사이트s like the Coherent Monaco provide the flexibility demanded by these applications thanks to the all-fiber design format. The resulting high average power, high energy 안전한 바카라 사이트 requirements, and the need to switch the stimulation beam on a sub-millisecond timescale presents a specific challenge for incumbent Pockels cell technology. To this end, AOM technology has been fully integrated into Monaco, for exquisite pulse control, simplified microscope design, and increased imaging system reliability.